Project Summary Development of the retina begins with the specification of retinal progenitor cells in the anterior neuroectoderm. The first morphological sign of this specification is the formation of two lateral grooves in the anterior neuroectoderm called the optic sulci. The cells of the optic sulci evaginate and form the optic vesicle. The distal portion of the optic vesicle will form the neuroretina and retina pigment epithelium, while the proximal portion will develop into the optic stalk. Our long-term goal is to identify developmental steps and molecular events necessary for the formation of the retina. The primary focus of our research is the role and mechanism of action of the homeobox gene Rx, a gene that appears to be at the top of the vertebrate retina-forming gene network. Rx encodes a transcription factor and mutations in this gene cause abnormal retinal development in all vertebrates investigated to date. The absence of Rx function leads to the absence of eye formation in mice and humans. We have shown previously that Rx activity is required cell-autonomously for the formation of the neuroretina, retina pigment epithelium and the distal optic stalk. How Rx functions at the molecular level is not yet fully understood. It is not known which proteins interact with Rx and which genes mediate its function. It is the goal of this project to determine the mode of action of Rx during retinal development. To better understand how Rx regulates retinal development and why mutations in this gene lead to such dramatic eye phenotypes, in Specific Aim 1 we will identify proteins that physically interact with Rx during retinal formation using genetically modified mice. In Specific Aim 2, we will identify direct target genes of Rx by chromatin immunoprecipitation combined with sequencing. This research will lead to a better understanding of retinal development, as this gene regulates the earliest critical steps in retinal formation. Furthermore, since mutations in this gene cause abnormal vertebrate eye development, this research will lead to the better diagnosis and treatment of diseases of the eye in which the components of the Rx regulatory network are mutated.